HVEM and nectin-1 are the major mediators of herpes simplex virus 1 (HSV-1) entry into human conjunctival epithelium

PURPOSE. The human conjunctiva is a natural target for herpes simplex virus (HSV)-1 infection. The goals of this study were to investigate the cellular and molecular mechanisms of HSV-1 entry into human conjunctival epithelial (HCjE) cells. Specific features of entry studied included the method of initial viral binding to cells, pH dependency, and expression and usage of specific HSV-1 entry receptors. METHODS. To observe HSV-1 initial binding, live cell imaging was performed on HSV-1-infected HCjE cells. Reporter HSV-1 virions expressing beta-galactosidase were used to determine entry of wild-type HSV-1(KOS) and a mutant, HSV-1(KOS) Rid1, into HCjE cells. HSV-1 replication in HCjE cells was determined by plaque assays. Lysosomotropic agents were used to determine whether viral entry was pH dependent. Reverse transcription (RT)-PCR, flow cytometry, and immunohistochemistry were used to determine the expression of receptors. Receptorspecific siRNAs were used to define the role of individual entry receptors. RESULTS. HSV-1 virions attach to filopodia present on HCjE cells and use them to reach the cell body for entry. Cultured HCjE cells demonstrate susceptibility to HSV-1 entry and form plaques confirming viral replication. Blocking vesicular acidification significantly reduces entry, implicating a pH-dependent mode of entry. Multiple assays confirm the expression of entry receptors nectin-1, HVEM, and 3-O-sulfated heparan sulfate (3-OS HS) on the HCjE cell membrane. Knocking down of gD receptors by siRNAs interference implicates nectin-1 and HVEM as the major mediators of entry. CONCLUSIONS. HSV-1 entry into HCjE cells is a pH-dependent process that is aided by targeted virus travel on filopodia. HCjE cells express all three major entry receptors, with nectin-1 and HVEM playing the predominant role in mediating entry.